Detergent-resistant membranes (DRMs) isolated from mammalian cells have been implicated in a diverse set of functions, ranging from intracellular sorting in polarized epithelial cells, to cell-surface signal transduction, to the structure of non-clathrin-coated plasma membrane pits called caveolae. Each of these roles will be examined, and a model explaining he structure of these membranes will be tested. Cholesterol-rich model membranes contain distinct domains; a novel phase called the liquid-ordered phase (Io) phase in addition to the liquid-crystalline (Ic) phase. Io phase lipids are packed more tightly than those in the Ic phase, but have more mobility than gel phase lipids. DRMs isolated from cells appear to be present in a phase similar to the Io phase. The long-term goal of this work is to determine how these domains function in cells. Many proteins that can be isolated from cells in DRMs are modified with saturated acyl chains (myristate and/or palmitate), that fit well into ordered domains. Three such proteins will be purified and incorporated into model membranes that contain Io phase domains; the caveolar coat protein caveolin, the Src- family kinase Yes, and the G protein Gi. The ability of these proteins to associate with DRMs from these liposomes in the absence of other proteins will be determined, as will the dependence of this behavior on protein acylation. Possible effects of Io phase domains on the function of these proteins will also be explored. These studies could have important health- related implications, as signaling proteins such as Src-family kinases can be oncogenic. Previous studies suggest that DRMs can be isolated from intracellular membranes as well as plasma membranes. DRMs will be isolated from the Golgi apparatus. Proteins found to be enriched in Golgi-derived DRMs may be important in sorting and trafficking along the secretory pathway, and will be further characterized. Complementary studies using cells and motel membranes will test our model that DRMs exist in cells as discrete domains. Recent results suggest that lipids with high acyl chain melting temperatures, such as sphingolipids, promote the formation of the Io phase in the presence of cholesterol. The lipid composition of DRMs from cells depleted of sphingolipid and/or cholesterol, and of model membranes that form DRMs, will be examined to test the correlation between formation of an Io-like phase and detergent-insolubility. Additional methods for examining the Io-like phase in model membranes and possibly in cell membranes will be used. Finally, the ability of the sphingolipid-poor inner leaflet of the plasma membrane to form DRMs will be tested using inside-out erythrocyte ghosts.